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1975-1976 (l)

[00:00:00] So this is Book l, beginning July 8th, 1975, and going through into 1976, February or thereabouts. Starts off with sequencing from gel. “Try ‘staining,’” in quotes, “with Braunitzer III — under acidic solutions, and eluting with [a?] disulfonic acid [compon?]‑‑ bicyclic…” I don’t know what this is. Think this is Braunitzer’s reagent — is a [00:01:00] coupling reagent allowing the attachment of… So the book begins, on Tuesday, July 8th, page one, with a so-called — heading of “Sequencing from the gel. Try ‘staining,’” in quotes, “with Braunitzer III reagent.” Braunitzer III reagent is a naphthalenedisulfonic acid thioisocyanate, which can be used to couple two amino groups — and then try eluting from Amido Black-[stained?] gels with alkaline reagent. Because, of course, the disulfonic acids make the material quite soluble, or might [00:02:00] do so. That was the idea. And next, pages of talking about this but no particular comment on the results. What was tested and what was not tested, in various stages of the reactions, A, B, C, D being the products to be tested. (pause)

Try to label, again, beta‑2 microglobulin. And a column elution, quite nicely… A nice peak obtained. Fraction 2 is [00:03:00] approximately 1 microcurie per nanomole. A reference to Cindy Shinnick’s notebook here and her work, on page 9, September 8th, CS‑3, page 71 — and what she used — with chlorohydrate and ethylene diacrylate — to make the gel more crosslinked. Use of a crosslink to change the consistency of the gel. [00:04:00] Tests of the products, a test first, electrophoresis plus or minus carrier and autorad before and after staining — was the idea. (laughs) Some technical problem, on page 13. “The gel stuck badly to the new,” quote, “‘stretch’ transparent film, not [Saran?].” But the conclusion is, first, that the results show clearly that the electrophoresis with and without the carrier is the same [00:05:00] and that staining of this 125I beta‑2‑M, even without carrier, shows protein, at about a half to 1 microgram. And the autoradiograph is pointing over.

Here now is a beginning of attempts to directly label proteins with tritium. I remember the experiments. They involved an enormous amount of radioactive and, in fact, carrier-free tritium, which was done by B. Wessels, whose first name, I think, was Barry — Barry Wessels — here on page 14, Wednesday, [00:06:00] September 10. “Tritium-labeling sample form, Physical Sciences Laboratory,” at Stoughton, Wisconsin. Talking about what the sample is. “The sample was 200mg of 125I‑labeled beta‑2‑M,” in parentheses, “(pure).” And my investigator name, etc. And, “The tritium mixture was 100% tritium.” And it was exposed to — my recollection, was exposed to high-frequency radio frequency [00:07:00] microwave — material. And the idea was to exchange some of the hydrogen atoms in the molecule for tritium and then sequence the tritiated material — so direct sequencing of proteins. And I do remember these experiments. I don’t remember the outcome. But no doubt we’ll find that out.

Tritiation experiments, then, Wednesday, September 10th, page 15. “Barry will produce, in about 100 ml of 7‑molar guanidinium hydrocholoride, 1 mc of tritiated beta‑2‑M, about 200 mg, containing 100,000 cpm/mg of 125I.” And a pattern to do the experiment. And the following pages are [00:08:00] talking about what — product was from the column, etc. With, Thursday, September 14, “The material –” although some crossing out there — “5 mmc/mg, checked by Barry.” But some indication of different fractions containing late tritium, with a thought that the first column fractions were the real material and later was free 14C carbon. So where the 14C is coming into [00:09:00] the — is not clear. I think that probably should have been tritium, not carbon. So the following page shows the columns and the result. But… And then, page 24, a repeat of the experiment, again with Barry Wessel, and a notification of everything that was done.

With an experiment at 120 watts, on page 25, the high-discharge… It was called a high-discharge experiment. [00:10:00] And as might be expected, but not yet described in these results, the higher the discharge of the radio frequency, the more fragmented material one obtained. So the graph on page 25 looks to be showing two experiments, one with high watts and one with lower watts. And more fragments in the higher discharge. But it’s not specifically commented on. Just the graphs are shown. And another experiment — and no discharge control — showing the 125I, the tritium, looking at different positions. So [00:11:00] just returning to page 25, the 125I and the tritium, eluting one peak, followed by a peak of tritium, which is junk. And when the same experiment is done without any discharge, there is almost — or very little label comes off in the 125I peak but the tritiated counts come out in the free volume of the column. No watts, 10 microliters, page 29. So it was working as expected. And with the follow-up, then, on Thursday, September [00:12:00] 11, that the mid-column peak is fragments. And trit‑‑ following e‑‑

An experiment, on Friday, September 12th, big crosses through it — saying, “Too hot.” What that means is not very clear. It’s perhaps the number of counts that are coming off. A fresh column, with the conclusion that the tritiated water is in Tube 10. More experiments of that general type, September 15.

And [00:13:00] some conclusions being reached and some quite simply interpreted results, on page 38 and 39. “Peak recovery 60% of theory.” And tritiated material [looked?] slightly ahead of the 125I — but no immediate comment on that. And junk coming out at parts of the column. But there is the comment on the non-overlapping of the peaks. “The tritium peak and the 125I peak do not coincide. So either the starting material is badly damaged –” that is to say, 125I‑labeled material [00:14:00] is not equivalent to the cold carrier — “or the tritiation is accompanied by severe damage, unfolding perhaps, non-proportional loss,” etc., etc., various attempts to understand that.

And a control G‑25 column, on page 40 and 41, with the optical density being slightly misplaced relative to the 125I peak. “The result is hard to interpret. Repeat with a different preparation.” So, repeated on the following page, [00:15:00] but only the 125. But, “The results are the same as yesterday,” that — etc. (pause)

[00:16:00] All then to try to get direct radioactive sequencing working — which never… We got a publication eventually but the method wasn’t really very practical.

Here, on page 55, a quite nice — and page 54, a quite nice column result that’s shown, with Dave McKean’s G‑75 column and 4‑molar guanidinium hydrochloride with 0.1‑molar ammonium bicarbonate — with a fairly pretty column, with — to Barry [00:17:00] Wessel. Point-one-four microcuries per microgram, [uncorrected?] — the material eluting at the correct place but an enormous amount of damaged product, of the order of 23,000 counts, versus 1,000 counts at the peak of real material.

More columns, the same general type. [00:18:00] On pages 57, 58, 59, continuing more samples from Barry Wessels, as we struggled to get a useful product. Continuing then — some experiments were being done with myoglobin. Tritium and 125I came off really exactly, [00:19:00] on the column, together, on this experiment, Thursday, September 2nd. (long pause)

Sequence [1095?], on page 69. [00:20:00] “Sequenced OK. Terrific.” So this is the comment, then. Etc. This… So let’s go back a little bit. Probably material is September 2nd material, in which the tritium label and the 125I label coincided very nicely — [00:21:00] and which is material prepared by Cindy Shinnick. “1.8 mgs of CS‑3, p. 39, Pool 2.” And we’re talking about page 67. That material, then, was used in sequenator run 1094 and “Sequenced OK” — “fairly OK” and the “fairly” is crossed out. And with a double exclamation mark, “Terrific!!” So the sequence was listed there, and with the amino acids fairly clearly… First position was isoleucine. Second position couldn’t be determined. Third was arginine — threonine, proline, and [00:22:00] isoleucine, as listed. And it goes on for 15 steps. So quite a nice sequence, isoleucine, question mark, arginine, threonine, proline, isoleucine, question mark, valine, etc. So there was a real sequence there, which is clearly tabulated, [in the?] numbers. And… I’ll pause that for a moment. (break in audio) So this result, the “Sequenced OK. Terrific!!” showing the sequence of beta‑2 microglobulin, was obtained. So that’s a really g‑‑ needs a little clarification again. So let’s go back to page 65, Thursday, September 2nd. [00:23:00] Bulk of tritiated material was obtained — and used on a column. And the result showed that the 125I and the tritium eluted very well together in the column. A graph, on page 64. And this material is talked about on page 67. And then the run of the material is from Book l‑‑64. Page 64 column material is then sequenced, on page 69, in the sequenator and, as mentioned before, [00:24:00] gave a very good sequence — beta‑2 microglobulin.

This is probably typical of the runs which made up a paper that was published in August of 1977, in the journal TIBS, which, I don’t remember what that is. It’s not easily found on searching by usual search in… A usual literature search doesn’t reveal this paper, but, “Protein Sequencing with Radioisotopes,” by Ian Walker, Oliver Smithies, Bryan Ballou, Liz Freedlander, and Cindy Shinnick. And talks about [00:25:00] the labeling, Edman degradation, and amino acid identification, etc. — Edman degradation. And then separate the proteins by slab electrophoresis and elute the proteins from gel particles, in this ca‑‑ and couple with isothiocyanate to — [de‑attaching?] beads. This was the bead method — it’s being talked about, and in this particular case. But sequence 1094 is a sequenator result, not a result from the Sequemat. So the paper on — August 1967 is talking about sequencing — the Sequemat. [00:26:00] So there was a result of that type. Anyway, the result on page 69 is very satisfactory, because clear, good sequence, and beta‑2 microglobulin.

And then an experiment with myoglobin, on page 71. And this was a big confusion. This experiment is now with myoglobin, and showing co‑elution of the labels of 125I and tritium, quite a large amount, as usual, of fragmented material. “Conclusions: Still some impurity pieces. But they look different from the beta‑2 microglobulin. [00:27:00] Maybe it is from protein breakdown.” Slowly getting the idea that the radio-frequency labeling fragmented the protein. A sequenator check, sequencing sequenator #1101, with just a comment, “OK.”

And so here, sequenator 1102, the aim was to see if the end-terminal yield of beta‑2 microglobulin, intrinsically labeled, is the same as beta‑2 microglobulin, extrinsically labeled — so making it with labeled [00:28:00] amino acids versus labeling it with the tritiation method.

Continuing with that general type of question. Here, on Thursday, November 13, page 81, is an experiment with myoglobin tritiated ex‑‑ from Dave McKean. Indications of a clean trailing shoulder but still much junk. So not just beta‑2‑M does this. It’s with a myoglobin experiment. [00:29:00] A lot of breakdown. (pause) Continuing with these experiment, the general type. (pause)

Trying to convert counts to — instead of trying to [00:30:00] count directly, count by fluorography, so instilling into the chlorohydrate gel a fluorophore, DPO, in DMSO, on page 91. With a result, “First try very weak, when done this wet way.” And with a result, presumably, of this type on page 93, 43‑hour exposure at -70°. [00:31:00] (pause) Tested with a gel with tritiated bovine serum albumen and tritiated pepsin, from Dave McKean. Again with [00:32:00] this fluorography method.

And continuing with the same general type of experiment. Friday, December 5th, page 97, some results of 24‑hour exposure and six-day exposure. Rather dirty but bands are very heavily labeled, although not very sharp. Run with 740 volts per 20 centimeter, air-cooled gel. This was an aluminum lactate, chlorohydrate gel, although it doesn’t say so exac– [00:33:00] Continuing in this vein.

Now a rather big step here, on page 100, talking about RNA gels for the first time, in relation to Dennis Drayna, talking about, “Wear gloves” and “Acid-washed glassware,” etc., etc., “Carrier, tRNA from Sigma,” [and?] etc. And acrylamide gel buffer, TBE — or TBV, I call… [00:34:00] But nothing yet on the RNA, just comments on the procedure.

With some summary of the tritiated thymidine conclusions, on page 104, that an increase in time causes increased damage. And, “Cystamine/EtOH radically changed the pattern.” Of the guanidinium hydrocholoride. “And the product looks cleaner on the gel but is of lower specific activity” — and other comments of that general type.

And Friday, January 2nd, 1976, [00:35:00] with a comment, “December 29th, 1975. Decided to change to DNA work full-time.” So this is the time when I shifted from being a protein sequencing person to working with DNA, a very critical change in my motivations and experiments. So it begins on Friday, January 2nd, 1976, page 109. “Tritiated thymidine of DNA test, combined with starting techniques for cloning.” So it talks about growing up a Charon phage, which was [00:36:00] Fred Blattner’s lab project — or products. There were many of them. Plus 14C, to give quantitation. “Purify the DNA on cesium chloride and check on the electron microscope. Tritiate cesium chloride, sucrose” etc., etc., “Therefore learn, first, transfection method, with borrowed DNA, second, bulk preparation, third, cesium chloride and sucrose.” So beginning to work with bacteria again.

Talking about spheroplasts and the genotype of the strain, [00:37:00] KH802, which is derived from C600. And, “gal‑, met‑, rK‑, mK+,” etc., etc., “Su‑2+,” with a suppressor gene in there. And the medium used. Just a learning curve for working with material. Transfection considered, on page 113. “DNA,” etc., “Equal volume as spheroplast. 30° for about 10 minutes,” being sufficient to get transfection. And thinking about multiplicity of infection. “0.05 [00:38:00] mg/ml of lambda DNA equivalent to a multiplicity of infection of 0.1,” which would be linear up to about 0.4 MOI. Onto NZ plates, which became very familiar. Oh. KH02 from Bill W. So who would Bill W. be? Anyway, from him. Streaked on NZ plates, which have enough methionine to growing… Single colony grown up. It’s beginning.

And a scheme, on February 23rd, to [00:39:00] label. (pause) A rather unclear scheme talked about, on page 123. [00:40:00] To try to get the hemoglobin gene, it appears is the idea. “Use a Hp-containing phage, cut to the left of Hp.” So I’m thinking of using a phage that already contains a copy of a hemoglobin gene. And indeed, I recognize the name here, on page 125. Leslie Furlong talked about this. Leslie Furlong was the person who did the first DNA sequencing, that I was concerned with, as we shall see in a little while. She was a graduate student, from McGill, I believe, a postdoc maybe or… I don’t remember exactly. But she was a very [00:41:00] capable young lady.

So the buffer, [Hellings?] 28 buffer was made, with the formula back in f — page 143, Book f. And here was Leslie Furlong, chromosome 6 DNA. Where she got that from, I don’t know. But the comment. For sticky ending. At the bottom of the page, “[R1?] from L.F.,” from Leslie Furlong, “good stuff. 1 ml per 10 ml, 3 to 4 hour‑‑” Not comprehensible.

But [00:42:00] a good DNA gel, on page 126, showing what was expected here, with [U173?], a Charon 6 gene — bacteriophage, rather, duplication. Oh, and a map of the chromosome. And then a DNA gel, stained with cesium bromide, not a very good gel, altogether, although the markers are quite nice. So it’s probably the product. But a good plot of size for the different fractions. [00:43:00] The usual type of size-versus-migration plot. And the gel being quite standard, half percent [CECAM?], four times Hellings, six volts total, at 0.7 volts per centimeter, and ran slowly overnight, from 10:00 p.m. to 8:30 a.m. And as I say, quite a nice gel.

And continuing, a gel electrophoresis of the different materials and with the markers being shown, and the gel, on page 128. So these are what became very familiar gels, of Hellings [00:44:00] gels with ethidium bromide staining. And the images were clearly taken with a Polaroid. Then some comments, on page 131 at the bottom, “A better scheme for the experiment does not require pre-sticky-ending.”

More, still, (laughs) experiment — was on Bill Williams’s… Evidently, B.W. is Bill Williams. Was one of Fred’s postdocs, as I recollect later. We’ll check on that. So this is [00:45:00] continuing on page 133, Friday, March 12th, the first reference, as I remember, in the book so far to Bill Williams, who was a postdoc of Fred Blattner’s and whom took part in a rather remarkable experiment, which no doubt I’ll mention a bit later. But in case I don’t, it’s Bill Williams, Fred Blattner, and Oliver Smithies drank bacteria and measured their outcome in the feces, (laughter) in testing the safety of the Charon phages. It formed a cover, of a paper, in Science, that will no doubt be referred to. But anyway, this is Bill Williams for the first time, with a Charon phage, and learning how to [00:46:00] digest it in different ways. A control and HindIII digests and markers. And the markers, I don’t remember what they were. They were probably bacteriophage digested with EcoR1. But whatever. They were standard markers, that we were used to making. The conclusion of this particular experiment — (laughs) “Once again, can’t find the sticky ends.”

Sticky ends again, looking for them, on page 137. “Can’t find [00:47:00] 11% on any,” is a comment. From Dave Moore, this material was obtained. “Try less salt.” More gels. Half percent Hellings [for?] buffer for the gel but different digests. “Repeat at a lower salt concentration,” for the dialysis of the [blood?]. Different ways. More sticky ends, March… Lambda wild-type tests, March 18th. So this sticky-end procedure was being tried. Try to understand what it was and [as we?]– For example, on page 146, it says, “This result shows that sticky ends were present but the 55° 2‑molar salt results in some loss. [And?] more results in more lo– Crazy!” exclamation mark. As this book wanders to its end.

So, page 150, it’s talking about Charon 3 and a future Charon 6, a [nuclear?] cloning idea, etc. So I’m going to have to find out what the sticky-end idea was. But [00:49:00] it didn’t survive in my memory, so it couldn’t eventually have been very useful. A new scheme is talked about on page 151, the last useful page in this book, a new scheme based on an earlier idea of mine, plus Rabbitts, with Charon 3. Completely uncomprehensible, at this point. And that ends this book. I’ll try to find out — [00:49:47]